This&#160;research was supported by NIH 1 U01 HL097973, NHLBI 5 R01 HL116511-02, UCSF-CTSI Grant Number UL1 TR000004, the Robert Wood Johnson Health and Society Scholars Program, and the Lisa and John Pritzker Family Foundation. The authors also wish to acknowledge Michelle Stephens for her assistance with scoring the ANS data as well as Vanessa Tearnan, Marialma Gonzales-Cruz, Yurivia Cervantes, Amy Engler, Stephanie Grover and Karen Jones-Mason for their assistance in collecting the ANS data, and Michael Coccia for his help with the data. We are also thankful to the families for their generous participation in this research and to the volunteers who helped us to test and refine this protocol, including the first author&#8217;s two children. We want to acknowledge our mentors who taught us about measuring ANS and why measuring ANS responsivity is meaningful in young children: W. Thomas Boyce, Gary Bernston, and Dave Lozano.

<ol>
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The authors declare that they have no competing financial interests.

Este estudio reveló que, dentro de una muestra de niños de 18 meses de edad, descansando estandarizada y tareas de desafío diseñadas para obtener respuestas de una serie de dominios (sobresalto, sensorial, social, descanso) dio lugar a una serie de respuestas del SNA. Las respuestas del SNA medios encontrados en estas condiciones de reposo y de desafío fueron similares a los valores reportados por otros ANS que han medido la capacidad de respuesta ANS con niños pequeños que utilizan tareas similares 8,11,27,...

Las diferencias individuales en el sistema nervioso autónomo de los niños (ANS) reactividad juegan un papel importante en el desarrollo y mantenimiento de los problemas de salud física y mental de 12-16. Un creciente cuerpo de evidencia revela que las diferencias individuales en las medidas de ANS en reposo y la reactividad son predictivos de las variaciones en los niños de psicopatología interiorizada y exteriorizada 4,9,17-19 y 7,20 salud física. Además de estos efectos directos o principales de funcionamiento ANS, una cantidad acumulada de estudios han encontrado que la reactividad de los niños puede interactuar con factores de riesgo contextuales, como los eventos adversos o conflictos de pareja, para moderar los efectos sobre el bienestar y la salud de los niños (véase la referencia 21 para una revisión). A pesar de esta evidencia creciente de que la reactividad autonómica de los niños juega un papel en la salud en todo el ciclo de vida y el interés que acompaña en estos procesos, hayse necesitan investigaciones adicionales para examinar el desarrollo del funcionamiento ANS en niños menores de tres años de edad. El SNA se compone de dos ramas, los sistemas nerviosos simpático y parasimpático. Regula la función de los órganos internos (por ejemplo, ritmo cardíaco, la respiración, la digestión y la excitación sexual), en gran parte a través de mecanismos inconscientes, en respuesta a las experiencias cotidianas y adversos 1. El sistema nervioso parasimpático (SNP) es el sistema de &#39;restaurador descanso y&#39; que mantiene una frecuencia cardíaca en reposo baja y el estado de restauración cuando se duerme o se relaja el sistema nervioso simpático (SNS) es el sistema de &quot;lucha o huida&quot; que responde a situaciones de emergencia o amenaza situaciones mediante la aceleración de uno de la frecuencia cardíaca 23. arritmia sinusal respiratoria (RSA) es un índice fiable de la influencia PNS sobre el funcionamiento cardiaco, y el período de pre-eyección (PEP) es un índice fiable de la influencia del SNS en el cochediac funcionamiento. Tanto RSA y PEP se han encontrado para ser medidas válidas mediante el bloqueo farmacológico experimental en muestras de adultos 24,25. Más recientemente, este trabajo se ha extendido a los niños y adolescentes muestras, estableciendo además RSA y PEP como medidas válidas y fiables de la actividad del SNA lo largo del desarrollo 8,26,27. Mediciones de cardiografía de impedancia fiables (PEP) utilizando electrodos de banda y punto se han utilizado durante un tiempo considerable en muestras de adultos 28. Más recientemente, las técnicas de medición y de análisis en paralelo también se han demostrado ser fiable y válido en muestras de niños 11,29,30, aunque colección de medidas de PEP en los niños pequeños es raro. Hay una gran cantidad existente de literatura sobre PNS que funcionan en los lactantes y niños pequeños, incluyendo el tono vagal y las medidas de la arritmia sinusal respiratoria. Hay menos estudios del SNS utilizando PEP, y muy pocos que examina simultáneamente elsistemas nerviosos simpático y parasimpático, lo cual es fundamental para la construcción de comprensión de su funcionamiento altamente integrado. De los pocos estudios de PEP de los niños pequeños, la mayoría encuentra que sus protocolos no producen cambios significativos en el nivel del grupo de PEP en respuesta a los desafíos 8,31. Los estudios han demostrado que existe estabilidad en descansando medidas PNS a partir de la infancia y tienen relaciones con el temperamento, el comportamiento y la salud, 13,16,26,32,33, pero hay una comprensión limitada de responder SNS y su estabilidad en el tiempo, relaciones con el desarrollo, y los factores que determinan que su trayectoria de desarrollo. Respuestas del SNP se han atribuido principalmente a la participación social y 16 respuestas SNS propuesta sobre la &quot;lucha o huida&quot; las respuestas 1, así como la sensibilidad recompensa (véase la referencia 34 para una revisión). En los niños pequeños, es un reto para simular retos específicos en un LaboratoRY y hacerlos participar debido a su capacidad de atención. También hay retos de medición para los niños que pueden experimentar incomodidad con los siete electrodos puntuales necesarios para medir PNS y SNS simultáneamente. Además, dentro de los campos de la ciencia del desarrollo, hay pocas normas sobre cómo obtener &quot;la reactividad al estrés&quot; suficientemente respetando al mismo tiempo las necesidades de desarrollo de los niños y la ética en el trabajo con los niños. En este artículo se presenta información sobre la administración y calificación de un protocolo estandarizado compuesto por condiciones de reposo y desafiantes diseñadas para obtener respuestas del sistema nervioso simpático y parasimpático de los niños de 18 meses de edad. Reactividad ANS es típicamente conceptualizado como la respuesta fisiológica a un estímulo externo discreto con respecto a una comparación o estado de reposo, que varía a través de los organismos individuales 22. El examen de la etiología de la vida temprana mental y física queproblemas Alth ha llevado a los científicos a ser particularmente interesados ​​en la comprensión de la reactividad de los niños a situaciones que evocan adaptativo de responder o pueden considerarse como retos estresantes. La preponderancia de la literatura se refiere a este fenómeno como &quot;la reactividad al estrés&quot;, a pesar de encontrarse con estímulos desafiantes tiene el potencial de provocar respuestas en una amplia gama de dominios. Por lo tanto, el protocolo descrito a continuación fue diseñado para obtener respuestas del SNA a través de múltiples dominios, incluyendo dos formas de reposo (mientras se escucha una canción de cuna calmantes y mientras se ve una calma, vídeo neutro) y tres desafíos apropiadas para el desarrollo de los niños de 18 meses: la anticipación / sobresalto (Jack-in-the-box), sensoriales (zumo de limón), y socio-emocional (escuchar un grito bebé enfermo). El protocolo fue adaptado de nuestros protocolos existentes para el mes 12 niños de 8 y 11 niños de 3-5 años, con el fin de hacer que el desarrollo desafiante, con la participación y tolerable para los niños (18-21 Meses). Aquí presentamos el Protocolo Desafíos del Desarrollo (DCP) y ANS datos de los 18 meses de edad visita de estudio del estrés, alimentación y Desarrollo Temprano (SEED) Estudio de las mujeres embarazadas y sus crías. maternas participantes con embarazos sanos fueron reclutados durante 2013-14 para un estudio de la ganancia de peso durante la gestación y el estrés prenatal y tenían sobrepeso, de bajos ingresos, y racial y étnicamente diversa. Se obtuvo el consentimiento informado de los padres para el estudio de la descendencia de ella justo después del nacimiento y otra vez antes del inicio de la recogida de datos para el estudio reportado aquí, cuando los hijos eran de 18-21 meses de edad durante 2014-2015.

<table>
	<tbody>
		<tr>
			<td>ANS equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Laptop and power cord&#160;</td>
			<td>Dell</td>
			<td>60-0107-3.0</td>
			<td>Mindware BioLab software requires a PC with 2 USB ports</td>
		</tr>
		<tr>
			<td>ANS data collection software</td>
			<td>Mindware</td>
			<td>contact company</td>
			<td>BioLab acquisition program</td>
		</tr>
		<tr>
			<td>ANS data acquisition unit and cords</td>
			<td>Mindware</td>
			<td>50-3711-08</td>
			<td>BioNex 8-slot Chassis</td>
		</tr>
		<tr>
			<td>Snap leads</td>
			<td>Mindware</td>
			<td>93-0498-00</td>
			<td>2 white, 2 red, 2 brown, 1 black</td>
		</tr>
		<tr>
			<td>Spot electrodes</td>
			<td>Kendall</td>
			<td>31112496</td>
			<td>130 model; 7 for each assessment + extras-to be sealed in a ziplock bag at all times)</td>
		</tr>
		<tr>
			<td>ANS scoring/editing software</td>
			<td>Mindware</td>
			<td>contact company</td>
			<td>HRV Analysis, version 3.1.0F and IMP Analysis, version 3.1.0I</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Protocol equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>tablet computer</td>
			<td>Apple</td>
			<td></td>
			<td>iPad; for playing audio and video clips</td>
		</tr>
		<tr>
			<td>portable speaker</td>
			<td>Polariod</td>
			<td>PBT510</td>
			<td></td>
		</tr>
		<tr>
			<td>Jack in the Box</td>
			<td>Schylling</td>
			<td>SMJB</td>
			<td>Sock Monkey theme</td>
		</tr>
		<tr>
			<td>Lemon juice</td>
			<td>ReaLemon</td>
			<td>98077</td>
			<td>0.14oz shelf-stable packets</td>
		</tr>
		<tr>
			<td>Pasture pipettes</td>
			<td>Grainger</td>
			<td>6FAV9</td>
			<td>1 ml size, disposable</td>
		</tr>
		<tr>
			<td>Portion cups</td>
			<td>Dart Solo Con</td>
			<td>250PC</td>
			<td>2.5 oz, disposable, plastic</td>
		</tr>
		<tr>
			<td>audio files of baby cry</td>
			<td>Alkon lab</td>
			<td></td>
			<td>30 second clip recorded in a neonatal intensive care unit</td>
		</tr>
		<tr>
			<td>audio file of baseline lullaby</td>
			<td>Rockabye Baby Music</td>
			<td></td>
			<td>60 seconds of &#34;Across the Universe&#34; on &#34;Rockabye Baby Lullaby Renditions of the Beatles&#34; CD</td>
		</tr>
		<tr>
			<td>audio file of final lullaby</td>
			<td>Rockabye Baby Music</td>
			<td></td>
			<td>60 seconds of &#34;Here Comes the Sun&#34; on &#34;Rockabye Baby Lullaby Renditions of the Beatles&#34; CD</td>
		</tr>
		<tr>
			<td>video file of neutral video</td>
			<td></td>
			<td></td>
			<td>2 minutes of Baby Einstein video</td>
		</tr>
		<tr>
			<td>Retractable measuring tape</td>
			<td>Rollfix</td>
			<td>TM04</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Miscellaneous materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Surge protector</td>
			<td></td>
			<td></td>
			<td>to plug in all electronics</td>
		</tr>
		<tr>
			<td>2-prong adapter for surge protector</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Video camera</td>
			<td>Sony</td>
			<td></td>
			<td>Handycam HDR-CX440</td>
		</tr>
		<tr>
			<td>Video Encoder, power and input cords</td>
			<td>Mindware</td>
			<td>50-8614-01</td>
			<td>optional; to sync video and ANS data files</td>
		</tr>
		<tr>
			<td>Baby wipes and gauze</td>
			<td></td>
			<td></td>
			<td>for cleaning and drying oily skin</td>
		</tr>
		<tr>
			<td>Toys</td>
			<td></td>
			<td></td>
			<td>to distract children during assessment</td>
		</tr>
		<tr>
			<td>Visual aids</td>
			<td></td>
			<td></td>
			<td>pictures of child with electrodes on, description of protocol to show participants</td>
		</tr>
		<tr>
			<td>Script of protocol for research assistants</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Charging cords for tablet and speaker</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Doll and reinforcer</td>
			<td></td>
			<td></td>
			<td>for demonstration if necessary</td>
		</tr>
	</tbody>
</table>

measuring cardiac autonomic nervous system (ans) activity in toddlers - resting and developmental challenges

El sistema nervioso autónomo (ANS) consta de dos ramas, los sistemas nerviosos simpático y parasimpático, y controla la función de los órganos internos (por ejemplo, el ritmo cardíaco, la respiración, la digestión) y responde a todos los días y las experiencias adversas 1. Se han encontrado medidas ANS en los niños estar relacionada con problemas de comportamiento, la regulación emocional y la salud 2-7. Por lo tanto, la comprensión de los factores que afectan el desarrollo ANS durante la primera infancia es importante. Ambas ramas del SNA afectan a las respuestas cardiovasculares de los niños pequeños a los estímulos y se han medido de forma no invasiva, a través de los equipos de vigilancia externa, utilizando medidas válidas y fiables de cambio fisiológico 8-11. Sin embargo, hay pocos estudios de los niños muy jóvenes con medidas simultáneas de los sistemas nerviosos simpático y parasimpático, lo que limita la comprensión del funcionamiento integrado de los dos sistemas. Además, el Majoridad de los estudios de los niños pequeños de informes existentes sobre las medidas de descanso ANS bebés o su reactividad al comúnmente utilizado paradigmas de interacción madre-hijo, y menos se sabe acerca de ANS reactividad a otras condiciones difíciles. Presentamos un diseño del estudio y protocolo estandarizado para la evaluación no invasiva y rápida del control autonómico cardiaco en niños de 18 meses de edad. Se describen métodos para la monitorización continua de las ramas simpático y parasimpático del SNA en condiciones de reposo y de desafío durante una visita al hogar o en el laboratorio y proporcionar resultados descriptivos de nuestra muestra de 140 niños con diversidad étnica utilizando el equipo y el software validado de puntuación. Los resultados revelaron que este protocolo puede producir una serie de respuestas fisiológicas al reposo y condiciones desafiantes del desarrollo, tal como se indica por los cambios en la frecuencia cardíaca y los índices de actividad simpático y parasimpático. Los individuos demostraron la variabilidad en los niveles de reposo, responses a los retos, y la reactividad desafío, que proporciona evidencia adicional de que este protocolo es útil para el examen de ANS diferencias individuales para los niños.

El estudio SEED inscrito 162 parejas madre-hijo (37% afroamericanos, 30% latinas; 16% blancos, 17% o más multirraciales). Para la visita 18 meses, hemos completado la evaluación en persona con 140 niños (87% de la muestra incluida); 6 participantes se alejaron, y el resto de participantes eran inalcanzables o no está disponible para esta visita. La tasa de rechazo de ANS componente de recopilación de datos de la visita fue 3/140 (2%) para las madres disponibles para esta visita. Tres visitas se llevaron a cabo desp...

Watch this Scientific Journal Video about Measuring Cardiac Autonomic Nervous System ANS Activity in Toddlers - Resting and Developmental Challenges at JoVE.com

Measuring Cardiac Autonomic Nervous System ANS Activity in Toddlers - Resting and Developmental Challenges

Se describen los métodos para la monitorización continua del sistema nervioso autónomo en condiciones de reposo y de desafío con niños de 18 meses de edad. Los resultados revelaron que este protocolo puede producir respuestas fisiológicas significativas en las dos ramas del sistema nervioso autónomo y provocar significativa variabilidad individual en los patrones de respuestas.

La medición del sistema nervioso autónomo cardíaco (ANS) La actividad en los niños pequeños - Descansar y Desafíos del Desarrollo

The autonomic nervous system (ANS) consists of two branches, the parasympathetic and sympathetic nervous systems, and controls the function of internal ...

measuring-cardiac-autonomic-nervous-system-ans-activity-toddlers

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Unit 1

Measuring Cardiac Autonomic Nervous System (ANS) Activity in Toddlers - Resting and Developmental Challenges

Biopsychology

The Autonomic Nervous System

SCIENTISTS IN ACTION

15.2K vistas.  University of California, San Francisco.  Se describen los métodos para la monitorización continua del sistema nervioso autónomo en condiciones de reposo y de desafío con niños de 18 meses de edad. Los resultados revelaron que este protocolo puede producir respuestas fisiológicas significativas en las dos ramas del sistema nervioso autónomo y provocar significativa variabilidad individual en los patrones de respuestas. 

Video: Measuring Cardiac Autonomic Nervous System ANS Activity in Toddlers - Resting and Developmental Challenges

Quantitative Autonomic Testing

Disorders associated with dysfunction of autonomic nervous system are quite common yet frequently unrecognized. Quantitative autonomic testing can be invaluable tool for evaluation of these disorders, both in clinic and research. There are number of autonomic tests, however, only few were validated clinically or are quantitative. Here, fully quantitative and clinically validated protocol for testing of autonomic functions is presented. As a bare minimum the clinical autonomic laboratory should have a tilt table, ECG monitor, continuous noninvasive blood pressure monitor, respiratory monitor and a mean for evaluation of sudomotor domain. The software for recording and evaluation of autonomic tests is critical for correct evaluation of data. The presented protocol evaluates 3 major autonomic domains: cardiovagal, adrenergic and sudomotor. The tests include deep breathing, Valsalva maneuver, head-up tilt, and quantitative sudomotor axon test (QSART). The severity and distribution of dysautonomia is quantitated using Composite Autonomic Severity Scores (CASS). Detailed protocol is provided highlighting essential aspects of testing with emphasis on proper data acquisition, obtaining the relevant parameters and unbiased evaluation of autonomic signals. The normative data and CASS algorithm for interpretation of results are provided as well.

Standardized, comprehensive and fully quantitative testing of autonomic functions is described. The autonomic tests consist of evaluation of all three major autonomic domains including cardiovagal, adrenergic and sudomotor. The severity and distribution of dysautonomia is quantitated using Composite Autonomic Severity Scores.

Prueba Autonómica cuantitativa

Disorders associated with dysfunction of autonomic nervous system are quite common yet frequently unrecognized. Quantitative autonomic testing can be ...

Investigación

Medicina

Measuring Cardiac Autonomic Nervous System (ANS) Activity in Children

The autonomic nervous system (ANS) controls mainly automatic bodily functions that are engaged in homeostasis, like heart rate, digestion, respiratory rate, salivation, perspiration and renal function. The ANS has two main branches: the sympathetic nervous system, preparing the human body for action in times of danger and stress, and the parasympathetic nervous system, which regulates the resting state of the body.
ANS activity can be measured invasively, for instance by radiotracer techniques or microelectrode recording from superficial nerves, or it can be measured non-invasively by using changes in an organ's response as a proxy for changes in ANS activity, for instance of the sweat glands or the heart. Invasive measurements have the highest validity but are very poorly feasible in large scale samples where non-invasive measures are the preferred approach. Autonomic effects on the heart can be reliably quantified by the recording of the electrocardiogram (ECG) in combination with the impedance cardiogram (ICG), which reflects the changes in thorax impedance in response to respiration and the ejection of blood from the ventricle into the aorta. From the respiration and ECG signals, respiratory sinus arrhythmia can be extracted as a measure of cardiac parasympathetic control. From the ECG and the left ventricular ejection signals, the preejection period can be extracted as a measure of cardiac sympathetic control. ECG and ICG recording is mostly done in laboratory settings. However, having the subjects report to a laboratory greatly reduces ecological validity, is not always doable in large scale epidemiological studies, and can be intimidating for young children. An ambulatory device for ECG and ICG simultaneously resolves these three problems.
Here, we present a study design for a minimally invasive and rapid assessment of cardiac autonomic control in children, using a validated ambulatory device 1-5, the VU University Ambulatory Monitoring System (VU-AMS, Amsterdam, the Netherlands,<a href="http://www.vu-ams.nl" target="_blank"> www.vu-ams.nl</a>).

Measuring Cardiac Autonomic Nervous System ANS Activity in Children

Measurement of autonomic nervous system activity usually confines the researcher and participant to the laboratory, which may provide an intimidating environment to children. The VU University Ambulatory Monitoring System (VU-AMS) device can record cardiac autonomic control in any setting. The VU-AMS proved very amenable to testing in children.

Sistema de medición de actividad nerviosa autonómica cardiaca (ANS) en los niños

The autonomic nervous system (ANS) controls mainly automatic bodily functions that are engaged in homeostasis, like heart rate, digestion, respiratory ...

Carotid Artery Infusions for Pharmacokinetic and Pharmacodynamic Analysis of Taxanes in Mice

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study model. As the use of mouse models are often a vital step in preclinical drug discovery and drug development1-8, it is necessary to design a system to introduce drugs into mice in a uniform, reproducible manner. Ideally, the system should permit the collection of blood samples at regular intervals over a set time course. The ability to measure drug concentrations by mass-spectrometry, has allowed investigators to follow the changes in plasma drug levels over time in individual mice1, 9, 10. In this study, paclitaxel was introduced into transgenic mice as a continuous arterial infusion over three hours, while blood samples were simultaneously taken by retro-orbital bleeds at set time points. Carotid artery infusions are a potential alternative to jugular vein infusions, when factors such as mammary tumors or other obstructions make jugular infusions impractical. Using this technique, paclitaxel concentrations in plasma and tissue achieved similar levels as compared to jugular infusion. In this tutorial, we will demonstrate how to successfully catheterize the carotid artery by preparing an optimized catheter for the individual mouse model, then show how to insert and secure the catheter into the mouse carotid artery, thread the end of the catheter out through the back of the mouse&#8217;s neck, and hook the mouse to a pump to deliver a controlled rate of drug influx. Multiple low volume retro-orbital bleeds allow for analysis of plasma drug concentrations over time.

This method was developed with the goal of delivering a steady drug solution via the carotid artery, to assess the pharmacokinetics of novel drugs in mouse models.

Infusiones de la arteria carótida para farmacocinéticas y farmacodinámicas Análisis de taxanos en ratones

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study ...

Electrophysiological Measurement of Noxious-evoked Brain Activity in Neonates Using a Flat-tip Probe Coupled to Electroencephalography

Pain is an unpleasant sensory and emotional experience. In non-verbal patients, it is very difficult to measure pain, even with pain assessment tools. Those tools are subjective or determine secondary physiological indicators which also have certain limitations particularly when exploring the effectiveness of analgesia. As cortical processing is essential for pain perception, brain activity measures may provide a useful approach to assess pain in infants. Here we present a method to assess nociception with electrophysiological brain activity recordings optimized for the use in newborn infants. To produce highly standardized and reproducible noxious stimuli we applied mechanical stimulation with a flat-tip probe, e.g., PinPrick, which is not skin-breaking and does not cause behavioral distress. The noxious-evoked potential allows the objective measurement of nociception in non-verbal patients. This method can be used in newborn infants as early as 34 weeks of gestational age. Moreover, it could be applied in different situations such as measuring the efficacy of analgesic or anesthetic drugs.

Measuring pain in non-verbal patients is a challenge. In this study we combine EEG recording with stimulation using a flat-tip probe to detect noxious-evoked brain activity in an objective manner.

Electrofisiológico medida de actividad cerebral evocados nocivos en neonatos con una sonda de punta plana juntada a la electroencefalografía

Pain is an unpleasant sensory and emotional experience. In non-verbal patients, it is very difficult to measure pain, even with pain assessment tools. ...

A Drosophila In Vivo Injury Model for Studying Neuroregeneration in the Peripheral and Central Nervous System

The regrowth capacity of damaged neurons governs neuroregeneration and functional recovery after nervous system trauma. Over the past few decades, various intrinsic and extrinsic inhibitory factors involved in the restriction of axon regeneration have been identified. However, simply removing these inhibitory cues is insufficient for successful regeneration, indicating the existence of additional regulatory machinery. Drosophila melanogaster, the fruit fly, shares evolutionarily conserved genes and signaling pathways with vertebrates, including humans. Combining the powerful genetic toolbox of flies with two-photon laser axotomy/dendriotomy, we describe here the Drosophila sensory neuron &#8211; dendritic arborization (da) neuron injury model as a platform for systematically screening for novel regeneration regulators. Briefly, this paradigm includes a) the preparation of larvae, b) lesion induction to dendrite(s) or axon(s) using a two-photon laser, c) live confocal imaging post-injury and d) data analysis. Our model enables highly reproducible injury of single labeled neurons, axons, and dendrites of well-defined neuronal subtypes, in both the peripheral and central nervous system.

A Drosophila In Vivo Injury Model for Studying Neuroregeneration in the Peripheral and Central Nervous System

Here, we present a protocol using the Drosophila sensory neuron - dendritic arborization (da) neuron injury model, which combines in vivo live imaging, two-photon laser axotomy/dendriotomy, and the powerful fly genetic toolbox, as a platform for screening potential promoters and inhibitors of neuroregeneration.

Un modelo de lesión de Drosophila en Vivo para el estudio de Neurorregeneración en el periférico y sistema nervioso Central

The regrowth capacity of damaged neurons governs neuroregeneration and functional recovery after nervous system trauma. Over the past few decades, various ...

Impact of Intracardiac Neurons on Cardiac Electrophysiology and Arrhythmogenesis in an Ex Vivo Langendorff System

Since its invention in the late 19th century, the Langendorff ex vivo heart perfusion system continues to be a relevant tool for studying a broad spectrum of physiological, biochemical, morphological, and pharmacological parameters in centrally denervated hearts. Here, we describe a setup for the modulation of the intracardiac autonomic nervous system and the assessment of its influence on basic electrophysiology, arrhythmogenesis, and cyclic adenosine monophosphate (cAMP) dynamics. The intracardiac autonomic nervous system is modulated by the mechanical dissection of atrial fat pads-in which murine ganglia are located mainly&#8212;or by the usage of global as well as targeted pharmacological interventions. An octapolar electrophysiological catheter is introduced into the right atrium and the right ventricle, and epicardial-placed multi-electrode arrays (MEA) for high-resolution mapping are used to determine cardiac electrophysiology and arrhythmogenesis. F&#246;rster resonance energy transfer (FRET) imaging is performed for the real-time monitoring of cAMP levels in different cardiac regions. Neuromorphology is studied by means of antibody-based staining of whole hearts using neuronal markers to guide the identification and modulation of specific targets of the intracardiac autonomic nervous system in the performed studies. The ex vivo Langendorff setup allows for a high number of reproducible experiments in a short time. Nevertheless, the partly open nature of the setup (e.g., during MEA measurements) makes constant temperature control difficult and should be kept to a minimum. This described method makes it possible to analyze and modulate the intracardiac autonomic nervous system in decentralized hearts.

Impact of Intracardiac Neurons on Cardiac Electrophysiology and Arrhythmogenesis in an Ex Vivo Langendorff System

Here, we present a protocol for the modulation of the intracardiac autonomic nervous system and the assessment of its influence on basic electrophysiology, arrhythmogenesis, and cAMP dynamics using an ex vivo Langendorff setup.

Impacto de las neuronas intracardiacas sobre la electrofisiología cardíaca y arritmogénesis en un sistema de Langendorff Ex Vivo

Since its invention in the late 19th century, the Langendorff ex vivo heart perfusion system continues to be a relevant tool for studying a broad spectrum ...

A Flow Cytometry-based Assay for Measuring Mitochondrial Membrane Potential in Cardiac Myocytes After Hypoxia/Reoxygenation

Timely and efficient reperfusion of the occluded coronary artery is the best strategy for decreasing myocardial infarct size in patients with a ST-segment elevated myocardial infarction. However, reperfusion per se can result in further cardiomyocyte death, a phenomenon known as reperfusion injury. The opening of the mitochondrial permeability transition pore (mPTP), with the decrease of the mitochondrial membrane potential (MMP), or mitochondrial depolarization, is universally recognized as the final step of reperfusion injury and is responsible for mitochondrial and cardiomyocyte death. JC-1 is a lipophilic cationic dye that accumulates in mitochondria depending on the value of MMP. The higher the MMP is, the more JC-1 accumulates in the mitochondria. The increasing amounts of JC-1 in mitochondria can be reflected by a fluorescence emission shift from green (~530 nm) to red (~590 nm). Therefore, the reduction of the red/green fluorescence intensity ratio can indicate the depolarization of mitochondria. Here, we take advantage of JC-1 to measure MMP, or the opening of mPTP in human cardiac myocytes after hypoxia/reoxygenation, detected by flow cytometry.

Here, we present a protocol that uses JC-1 dye to assess the mitochondrial membrane potential of cells after being exposed to hypoxia/reoxygenation with or without a protective agent.

Un análisis basado en la citometría de flujo para medir el potencial de membrana mitocondrial en miocitos cardiacos después de hipoxia/Reoxygenation

Timely and efficient reperfusion of the occluded coronary artery is the best strategy for decreasing myocardial infarct size in patients with a ST-segment ...

Semi-Minimal Invasive Method to Induce Myocardial Infarction in Rats and the Assessment of Cardiac Function by an Isolated Working Heart System

Myocardial infarction (MI) remains the main contributor to morbidity and mortality worldwide. Therefore, research on this topic is mandatory. An easily and highly reproducible MI induction procedure is required to obtain further insight and better understanding of the underlying pathological changes. This procedure can also be used to evaluate the effects or potency of new and promising treatments (as drugs or interventions) in acute MI, subsequent remodeling and heart failure (HF). After intubation and pre-operative preparation of the animal, an anesthetic protocol with isoflurane was performed, and the surgical procedure was conducted&#160;quickly. Using a minimally invasive approach, the left anterior descending artery (LAD) was located and occluded by a ligature. The occlusion can be performed acutely for subsequent reperfusion (ischemia/reperfusion injury). Alternatively, the vessel can be ligated permanently to investigate the development of chronic MI, remodeling or HF. Despite common pitfalls, the drop-out rates are minimal. Various treatments such as remote ischemic conditioning can be examined for their cardioprotective potential pre-, peri- and post-operatively. The post-operative recovery was quick as the anesthesia was precisely controlled and the duration of the operation was short. Post-operative analgesia was administered for three days. The minimally invasive procedure reduces the risk of infection and inflammation. Furthermore, it facilitates rapid recovery. The &#8220;working heart&#8221; measurements were performed ex vivo and enabled precise control of preload, afterload and flow. This procedure requires specific equipment and training for adequate performance. This manuscript provides a detailed step-by-step introduction for conducting these measurements.

This article presents an efficient method to perform myocardial ischemia and subsequent chronic reperfusion in rats using a minimally invasive approach. In addition, left ventricular hemodynamic function of rats is assessed by echocardiography and isolated working heart methods.&#160;

Método invasivo semiseminal para inducir el infarto de miocardio en ratas y la evaluación de la función cardíaca mediante un sistema cardíaco de trabajo aislado

Myocardial infarction (MI) remains the main contributor to morbidity and mortality worldwide. Therefore, research on this topic is mandatory. An easily ...

In vitro Assessment of Cardiac Reprogramming by Measuring Cardiac Specific Calcium Flux with a GCaMP3 Reporter

Cardiac reprogramming has become a potentially promising therapy to repair a damaged heart. By introducing multiple transcription factors, including Mef2c, Gata4, Tbx5 (MGT), fibroblasts can be reprogrammed into induced cardiomyocytes (iCMs). These iCMs, when generated in situ in an infarcted heart, integrate electrically and mechanically with the surrounding myocardium, leading to a reduction in scar size and an improvement in heart function. Because of the relatively low reprogramming efficiency, purity, and quality of the iCMs, characterization of iCMs remains a challenge. The currently used methods in this field, including flow cytometry, immunocytochemistry, and qPCR, mainly focus on cardiac-specific gene and protein expression but not on the functional maturation of iCMs. Triggered by action potentials, the opening of voltage-gated calcium channels in cardiomyocytes leads to a rapid influx of calcium into the cell. Therefore, quantifying the rate of calcium influx is a promising method to evaluate cardiomyocyte function. Here, the protocol introduces a method to evaluate iCM function by calcium (Ca2+) flux. An &#945;MHC-Cre/Rosa26A-Flox-Stop-Flox-GCaMP3 mouse strain was established by crossing Tg(Myh6-cre)1Jmk/J (referred to as Myh6-Cre below) with Gt(ROSA)26Sortm38(CAG-GCaMP3)Hze/J (referred to as Rosa26A-Flox-Stop-Flox-GCaMP3 below) mice. Neonatal cardiac fibroblasts (NCFs) from P0-P2 neonatal mice were isolated and cultured in vitro, and a polycistronic construction of MGT was introduced to NCFs, which led to their reprogramming to iCMs. Because only successfully reprogrammed iCMs will express GCaMP3 reporter, the functional maturation of iCMs can be visually assessed by Ca2+ flux with fluorescence microscopy. Compared with un-reprogrammed NCFs, NCF-iCMs showed significant calcium transient flux and spontaneous contraction, similar to CMs. This protocol describes in detail the mouse strain establishment, isolation and selection of neonatal mice hearts, NCF isolation, production of retrovirus for cardiac reprogramming, iCM induction, the evaluation of iCM Ca2+ flux using our reporter line, and related statistical analysis and data presentation. It is expected that the methods described here will provide a valuable platform to assess the functional maturation of iCMs for cardiac reprogramming studies.

We describe here, the establishment and application of an Tg(Myh6-cre)1Jmk/J /Gt(ROSA)26Sortm38(CAG-GCaMP3)Hze/J (referred to as &#945;MHC-Cre/Rosa26A-Flox-Stop-Flox-GCaMP3 below) mouse reporter line for cardiac reprogramming assessment. Neonatal cardiac fibroblasts (NCFs) isolated from the mouse strain are converted into induced cardiomyocytes (iCMs), allowing for convenient and efficient evaluation of reprogramming efficiency and functional maturation of iCMs via calcium (Ca2+) flux.

Evaluación in vitro de la reprogramación cardíaca mediante la medición del flujo de calcio específico cardíaco con un informador GCaMP3

Cardiac reprogramming has become a potentially promising therapy to repair a damaged heart. By introducing multiple transcription factors, including ...

Evaluación de la hipercoagulación sanguínea del plasma rico en EV

Determination of the Procoagulant Activity of Extracellular Vesicle (EV) Using EV-Activated Clotting Time (EV-ACT)

The role of extracellular vesicles (EV) in various diseases is gaining increased attention, particularly due to their potent procoagulant activity. However, there is an urgent need for a bedside test to assess the procoagulant activity of EV in clinical settings. This study proposes the use of thrombin activation time of EV-rich plasma as a measure of EV's procoagulant activity. Standardized procedures were employed to obtain sodium-citrated whole blood, followed by differential centrifugation to obtain EV-rich plasma. The EV-rich plasma and calcium chloride were added to the test cup, and the changes in viscoelasticity were monitored in real-time using an analyzer. The natural coagulation time of EV-rich plasma, referred to as EV-ACT, was determined. The results revealed a significant increase in EV-ACT when EV was removed from plasma obtained from healthy volunteers, while it significantly decreased when EV was enriched. Furthermore, EV-ACT was considerably shortened in human samples from preeclampsia, hip fracture, and lung cancer, indicating elevated levels of plasma EV and promotion of blood hypercoagulation. With its simple and rapid procedure, EV-ACT shows promise as a bedside test for evaluating coagulation function in patients with high plasma EV levels.

Autor destacado: Descifrando los trastornos de la coagulación en pacientes con lesiones cerebrales traumáticas 

This protocol investigates the use of extracellular vesicle (EV)-rich plasma as an indicator of the coagulative ability of EV. EV-rich plasma is obtained through a process of differential centrifugation and subsequent recalcification.

Determinación de la actividad procoagulante de la vesícula extracelular (VE) mediante el tiempo de coagulación activado por EV (EV-ACT)

The role of extracellular vesicles (EV) in various diseases is gaining increased attention, particularly due to their potent procoagulant activity. ...